Genetic basis of an elite wheat cultivar Guinong 29 with harmonious improvement between multiple diseases resistance and other comprehensive traits

Fungal diseases, such as powdery mildew and rusts, significantly affect the quality and yield of wheat. Pyramiding diverse types of resistance genes into cultivars represents the preferred strategy to combat these diseases. Moreover, achieving collaborative improvement between diseases resistance, abiotic stress, quality, and agronomic and yield traits is difficult in genetic breeding. In this study, the wheat cultivar, Guinong 29 (GN29), showed high resistance to powdery mildew and stripe rust at both seedling and adult plant stages, and was susceptible to leaf rust at the seedling stage but slow resistance at the adult-plant stage. Meanwhile, it has elite agronomic and yield traits, indicating promising coordination ability among multiple diseases resistance and other key breeding traits. To determine the genetic basis of these elite traits, GN29 was tested with 113 molecular markers for 98 genes associated with diseases resistance, stress tolerance, quality, and adaptability. The results indicated that two powdery mildew resistance (Pm) genes, Pm2 and Pm21, confirmed the outstanding resistance to powdery mildew through genetic analysis, marker detection, genomic in situ hybridization (GISH), non-denaturing fluorescence in situ hybridization (ND-FISH), and homology-based cloning; the stripe rust resistance (Yr) gene Yr26 and leaf rust resistance (Lr) genes Lr1 and Lr46 conferred the stripe rust and slow leaf rust resistance in GN29, respectively. Meanwhile, GN29 carries dwarfing genes Rht-B1b and Rht-D1a, vernalization genes vrn-A1, vrn-B1, vrn-D1, and vrn-B3, which were consistent with the phenotypic traits in dwarf characteristic and semi-winter property; carries genes Dreb1 and Ta-CRT for stress tolerance to drought, salinity, low temperature, and abscisic acid (ABA), suggesting that GN29 may also have elite stress-tolerance ability; and carries two low-molecular-weight glutenin subunit genes Glu-B3b and Glu-B3bef which contributed to high baking quality. This study not only elucidated the genetic basis of the elite traits in GN29 but also verified the capability for harmonious improvement in both multiple diseases resistance and other comprehensive traits, offering valuable information for breeding breakthrough-resistant cultivars.


Plant materials
The wheat cultivar GN29 was developed from a cross between the wheat cultivar Guinong 13 (GN13) and the wheat breeding line Guinong 21 (GN21) using marker-assisted selection (MAS) by Guizhou University and Guizhou Sub-center of the National Wheat Improvement Center and released in 2014.The wheat cultivar Ping'an 9 (PA9) was susceptible to powdery mildew and stripe rust and used as the susceptible parent to cross with GN29 to obtain the F 1 , F 2 , and F 2:3 populations for genetic and lineage analysis of Pm and Yr genes in GN29.The wheat cultivar Mingxian 169 (MX169) was susceptible to powdery mildew, stripe rust, and leaf rust and used as a susceptible check in multiple diseases resistance evaluation.Nineteen and fourteen wheat donors carrying known Pm genes and Yr genes, respectively, served as positive controls in the molecular marker detection and/ or homology-based cloning experiments (Supplementary Table S1).Donors of these Pm and Yr genes were provided by Prof. Hongxing Xu, Henan University, Kaifeng, China and Prof. Caixia Lan, Huazhong Agricultural University, Wuhan, China, respectively.

Resistance assessment to multiple wheat diseases
To assess the powdery mildew resistance, 31 single-spore-derived Bgt isolates with different virulence spectra, provided by Prof. Yilin Zhou, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, and Prof. Hongxing Xu, Henan University, were used to test the seedling reaction patterns of GN29 using MX169 as the susceptible control and nine resistant donors with known Pm genes as the resistant controls.Five seeds of each genotype were planted in trays (54 × 28 × 4.2 cm) with 128 cells (3.2 × 3.2 × 4.2 cm).When the seedlings grown to the two-leaf stage, they were inoculated with the fresh conidiospores previously developed on the MX169 seedlings.Seedlings in different trays were inoculated with the 31 Bgt isolates separately, and each tray was covered with a glass shroud to avoid cross-infection between different isolates.Several resistance stocks with documented Pm genes in production or with high resistance were used as resistant controls and MX169 was used as susceptible control.When the pustules were fully developed on the first leaves of MX169 seedlings, approximately 14-15 days after inoculation, infection types (ITs) for each plant were scored based on a 0-4 scale standard, of which ITs 0-2 were regarded as resistant and ITs 3 and 4 as susceptible 21 .All tests were repeated thrice to ensure data reliability.
At the adult stage, GN29 was inoculated with a mixture of all the Bgt isolates used in the seedling stage for three consecutive years (2018 to 2020) at Yantai University, Yantai City, Shandong Province, China (121.39′E, 37.52′ N).Sowing and inoculation methods were referred to our proven technique system 22 .Disease reaction at the adult stage was scored on a 0-9 scale, of which 0-4 was considered as resistant and 5-9 as susceptible 21 .Each plant was assessed twice.
For stripe rust resistance assessment, three Pst isolates, CYR32, CYR33, and CYR34 with different virulence spectra were used to test the seedling reaction patterns of GN29 using MX169 as the susceptible control.Assessments of adult plant stripe rust responses were conducted at Yantai University using a mixture of CYR32, CYR33, and CYR34.The sowing and inoculation methods were referred to the reported procedure 23 .The ITs at the seedling and adult plant stages were both scored based on a scale of 0-9, of which ITs ranging from 0 to 6 were classified as resistant, whereas ITs 7-9 as susceptible 24 .
For the leaf rust resistance assessment, mixed P. triticina pathotypes collected in Yantai, China were used to test the seedling and adult-stage reaction patterns of GN29 using MX169 as the susceptible control.The sowing and inoculation methods were referred to the reported procedure 18 .Infection types were scored according to the Stakman scale with moderate modification 25 .

Assessment of agronomic and yield performance
GN29 were planted at Guiyang city, Guizhou Province (26.57N, 106.71 E), China, area available for popularization and Langfang City, Hebei Province (39.53 N, 116.72 E), China, in a randomized complete block design with three replicates.The wheat cultivars Guinong 19 (GN19) and Yannong 999 (YN999) were used as controls in Guizhou and Langfang, respectively.Each cultivar was planted as a plot with three rows (length: 1.5 m; distance between rows: 0.25 m) and 30 seeds per row.Three plants in the middle of the two internal rows were sampled to evaluate the plant height (PH), spike numbers per plant (SNPP), spikelet numbers per spike (SNS), sterile spikelet numbers per spike (SSNS), kernel numbers per spike (KNS), and thousand-kernel weight (TKW).

Genetic analysis, molecular marker detection, and homology-based cloning of the Pm genes
To determine the inheritance of powdery mildew resistance in GN29 at the seedling stage, two Bgt isolates E09 (prevalent) and E18 (hypertoxic) were used to inoculate GN29 and PA9 and their F 1 hybrids, F 2 population, and F 2:3 families (30 seeds per F 2:3 family were selected) at the one-leaf stage.After phenotyping, goodness-of-fit was analyzed using the chi-square (χ 2 ) test to investigate deviations in the observed phenotypic data of the F 2 populations and F 2:3 families from the theoretically expected segregation ratios.
Total genomic DNA was isolated using the cetyltrimethylammonium bromide (CTAB) method from young leaves of the wheat seedlings 27 .To detect the Pm genes in GN29, 39 diagnostic/linked markers of 31 known Pm genes were used to genotype GN29, PA9, and 19 wheat donors with known Pm genes (Supplementary Tables S1  and S2).Polymorphic markers were genotyped in the corresponding F 2:3 families.
PCR amplification and visualization were performed as described in our lab 27 .PCR amplification was carried out in a 10 μL volume system, including 5 μL 2 × Taq Master Mix (Vazyme, China), 1 μL 50 ng/μL template DNA and 0.5 μL 10 μM/μL primers.The PCR amplification condition was set as follows: pre-denaturation at 94 °C for 5 min followed by 36 cycles of 94 °C for 30 s, 50-65 °C (depending on the specific primers) for 40 s, 72 °C for 40-120 s (depending on the target bands), finally, extension at 72 °C for 10 min and preservation at 25 °C.The PCR products were then separated on 8% non-denaturing polyacrylamide gels with a 29:1 ratio of acrylamide to bis-acrylamide or 1.5% agarose gel based on the size of the target bands 28,29 .
After confirming the presence of Pm genes in GN29, total RNA from the young leaves of GN29 were extracted using the Spectrum Plant Total RNA kit (Sigma-Aldrich, Shanghai, China) following the manufacturer's recommendations.The RNA samples were quantified by measuring the absorbance at 260 and 280 nm using a NanoDrop 1000 spectrophotometer (Thermo Scientific, Shanghai, China).High-quality RNA was treated with Promega DNase I for cDNA synthesis using Invitrogen SuperScript II reverse transcriptase, according to the manufacturer's guidelines.Based on the reports of the cloning of Pm2 30 and Pm21 31 , the full length of the homologous sequences of Pm2 and Pm21 were isolated.After obtaining the coding sequence (CDS) of Pm2 and Pm21, they were sequenced using Sanger sequencing and compared with those of Pm2 30 and Pm21 31 .

Cytogenetic analysis
Genomic in situ hybridization (GISH) was firstly performed to detect D. villosum chromatin in the GN29.Mitotic chromosomes of the root tip cells of GN29 were prepared and observed, as previously described 32 .The genomic DNA of D. villosum was labeled with fluorescein-12-dUTP as a probe to detect chromosomal fragments for GISH.After hybridization with probes, the chromosomes were counterstained with propidium iodide (PI) and mounted on Vectashield (Roche Co., Burlingame, CA, USA).Signals were examined under an Olympus BX60 epifluorescence microscope (Olympus Co., Tokyo, Japan).
To clearly determine the chromosome composition of GN29, we also performed non-denaturing fluorescence in situ hybridization (ND-FISH) to analyze mitotic chromosomes of the root tip cells.The probes in this study were Oligo-pSc199.2-1(green) and Oligo-pTa535-1 (red), and they were distributed as 5′ end-labeled with 6-carboxyfluorescein (FAM) and 6-carboxytetramethylrhodamine (TAMRA).

Molecular marker detection of the Yr and Lr genes
To determine the presence of Yr genes in GN29, 17 diagnostic/linked markers for known Yr genes were used to test GN29 and PA9, using 14 resistant donors with known Yr genes as controls (Supplementary Table S2).If the polymorphic band(s) of one Yr gene were detected in GN29 and not PA9, this Yr gene would most likely exist in GN29.To confirm this result, the polymorphic markers were also used to genotype the segregated populations of GN29 and PA9.
To detect the presence of Lr genes in GN29, a similar but simplified procedure was performed using 12 diagnostic/linked markers of known Lr genes by comparing the polymorphic band(s) in GN29 and PA9 but not by genotyping the segregation population (Supplementary Table S2).PCR amplification and product visualization were performed as described above [27][28][29] .

Agronomic and yield performance of GN29
When GN29 was planted in Guizhou Province, it showed comprehensively excellent performance for the investigated traits, including PH, SNPP, SNS, SSNS, KNS, and TKW (Table 1), no obvious disadvantages were detected.Compared with the famous wheat cultivar GN19, GN29 still has significant advantages in terms of SNPP, SSNS, and TKW.To investigate the adaptation of GN29 in other wheat production region, GN29 was also surveyed in Langfang City, Hebei Province.Compared to the performance in Guizhou Province, GN29 significantly increased SNPP but decreased TKW.Significant decrease in the PH was also observed (Fig. 1, Table 1).Although significant changes in agronomic and yield performance occurred in other distinct agroecological area, GN29 still showed better adaptation.Even compared to the famous wheat cultivar YN999, GN29 still showed satisfactory agronomic and yield performance in the Langfang region (Fig. 1, Table 1).

Evaluation and inheritance of the powdery mildew resistance in GN29
When tested with the Bgt isolate E09, GN29 showed hypersensitivity on the first leaves and can be regarded as immune with IT 0;, whereas PA9 showed abundant sporulation with > 80% of the first leaves covered with aerial hyphae and hence as highly susceptible with IT 4. The F 1 plants of GN29 × PA9 showed a similar reaction pattern to E09 as that to GN29 with an IT 0;, suggesting that the Pm gene(s) in GN29 displayed dominant inheritance.The F 2 population fitted the theoretical ratio of 15:1 for the segregation model of the two dominant genes (Table 2).
Another Bgt isolate E18 was also used to inoculate GN29, PA9, and their derived F 1 plants, F 2 populations, and F 2:3 families.Interestingly, a dominant monogenic segregation model was clearly observed using this highly virulent Bgt isolate, suggesting that one of the two Pm genes in GN29 was defeated by this Bgt isolate and the remaining Pm gene displayed dominant monogenic inheritance (Table 2).
To further assess seedling resistance to powdery mildew in GN29, it was inoculated with 31 Bgt isolates using several Pm gene donors in production or with a high breeding value as controls.The results showed that GN29 was immune to all the tested Bgt isolates (Fig. 2, Table 3).Compared to the other tested Pm genes, GN29 had broad spectrum resistance.At the adult stage, GN29 was also immune to the Bgt mixture in three consecutive years.Therefore, GN29 showed elite powdery mildew resistance at the whole growth stage.

Molecular detection and homology-based cloning of the Pm genes in GN29
To identify the Pm genes in GN29, 39 diagnostic/linked markers for 31 known Pm genes were selected to test GN29 and PA9 (Table 4).Only the diagnostic markers Pm2b-map-3 for Pm2 and MBH1 for Pm21 amplified the targeted bands of Pm2 and Pm21, respectively, in GN29 but not in PA9.To further verify the presence of Pm2 and Table 1.Agronomic and yield traits of Guinong 29 (GN29) grown in Guizhou and Hebei provinces of China using wheat cultivars Guinong 19 (GN19) and Yannong 999 (YN999) as controls, respectively.Values in the same column followed by the same letter were not significantly different based on the test of T test at P < 0.05.PH: plant height; SNPP: spike number per plant; SNS: spikelet number per spike; SSNS: sterile spikelet numbers per spike; KNS: kernel number per spike; TKW: thousand-kernel weight.As expected, MBH1 is co-segregated with the phenotype, suggesting the presence of Pm21 (Fig. 3).To confirm the existence pattern of Pm21, GISH analysis was carried out and showed that GN29 had a pair of alien chromosome arms of D. villosum (Fig. 4A).ND-FISH analysis further showed that the alien chromosome arms were 6VS in GN29 (Fig. 4B).The diagnostic marker Pm2b-map-3 of Pm2 was also used to genotype the susceptible plants of the F 2 population phenotyped by the Bgt isolate E09, and it co-segregated with the phenotype, suggesting the presence of Pm2.To clarify the allelic types of Pm2 and Pm21 in GN29, we cloned their homologous sequences.Following sequence alignment, the haplotypes in GN29 were confirmed as Pm2a and Pm21.

Evaluation and identification of stripe rust and leaf rust resistance in GN29
When tested with Pst isolates CYR32, CYR33, and CYR34 at the seedling stage, GN29 showed high resistance with IT 1.When tested with a mixture of these three Pst isolates at the adult plant stage, GN29 also showed high resistance ranging from IT1-2.For the leaf rust resistance assessment, GN29 was highly susceptible to the mixed Pt races; however, it showed slow reactance at the adult plant stage, suggesting that the slow leaf rust resistance gene may be involved in GN29.
To investigate the Yr gene(s) in GN29, 17 diagnostic/linked molecular markers for 14 known Yr genes were used to test GN29 (Table 4).The results showed that only the Yr26-linked marker WE173 could amplify the target band, suggesting that GN29 was most likely to carry Yr26.To further verify the presence of Yr26, WE173 was used Table 2. Segregation ratios of F 2 and F 2:3 generations of Guinong 29 (GN29) and Ping'an 9 (PA9) following inoculation with Blumeria graminis f. sp.tritici (Bgt) isolates E09 and E18 at the seedling stage.Values of χ 2 for statistical significance at P = 0.05 are 3.84 (df1) and 5.99 (df2); R: Resistant, S: Susceptible, HR: homozygous resistant, Seg: segregating, HS: homozygous susceptible.

Plants observed
Expected ratio Χ www.nature.com/scientificreports/ to genotype the F 2:3 families phenotyped by isolate CRY34.The result showed that WE173 was co-segregated with phenotypes, further confirming the presence of Yr26 (Fig. 5).A simple and similar procedure was also carried out to detect the Lr gene(s) in GN29 by analyzing the polymorphic band(s) in GN29 but not by genotyping the segregation population, suggesting that Lr1 and Lr46 may exist in GN29 (Supplementary Table S2).

Molecular identification of drought tolerance and preharvest sprouting resistance in GN29
To investigate the drought tolerance and preharvest sprouting resistance genes in GN29, six markers linked to two main drought tolerance genes and three markers linked to three main preharvest sprouting resistance genes were used to detect GN29 (Table 4).The results indicated that markers P18, P20, P21, P22, and P25 associated with the drought tolerance gene Dreb1, and DF/DR linked to Ta-CRT , successfully amplified target bands, suggesting that GN29 was most likely to carry Dreb1 and Ta-CRT , implying the potential drought tolerance of GN29 (Supplementary Table S2).However, marker detection results also indicated that GN29 had none of the three main preharvest sprouting resistance genes, TaAFP-Bb/TaAFP-Ba, Vp-1Bb/Vp-1Ba/Vp-1Bc, and Vp-1Ba/ Vp-1Bf, implying potential risks in pre-harvest sprouting (Supplementary Table S2).

Molecular identification of vernalization and dwarfing genes in GN29
To investigate the vernalization and dwarfing genes in GN29, nine markers linked to eight vernalization genes and five markers linked to five dwarfing genes were used to detect GN29.The results indicated that the markers BF-WR1 linked to Rht-B1b, DF-MR2 linked to Rht-D1a and gwm261 linked to Rht8 could amplify the target bands, suggesting that GN29 most likely carries the dwarfing genes Rht-B1b, Rht-D1a, and Rht8 (Table 4, Supplementary Table S2).The PH of GN29 planted in Guizhou Province did not correlate with the presence of Rht-B1b, Rht-D1a, and Rht8; however, the PH in Hebei Province was mostly affected by their presence.For the detection of vernalization genes, GN29 had four of the seven tested vernalization genes vrn-A1, Vrn-B1, vrn-D1, and Vrn-B3, hence, GN29 could be considered a semi-winter cultivar.The better adaptation in Guizhou and Hebei Provinces may be related to this factor.

Molecular identification of quality-related genes in GN29
To investigate the quality-related genes in GN29, 22 markers linked to 22 glutenin subunit genes were used to detect GN29.The result indicated that the markers SB2 linked to Glu-B3b and SB10 linked to Glu-B3bef could amplify the target bands, suggesting that GN29 was most likely to carry two low-molecular-weight glutenin subunit genes, Glu-B3b and Glu-B3bef, which contribute to the malleability of the dough and food processing quality (Table 4, Supplementary Table S2).We noticed that the elite high-molecular-weight glutenin subunit genes Dx5 and Dx10 were absent in GN29, which may affect the gluten strength of GN29 (Table 4, Supplementary Table S2).

Discussion
Wheat breeding involves the integration of elite traits from diverse donors into a unified genetic background.In this process, harmonious improvement between different traits is critical, particularly between multiple diseases resistance and other comprehensive traits 13 .For instance, it is difficult to pyramid large mumble of elite traits between multiple diseases resistance and stress tolerance, adaptation, quality, and high yield and better express all of them in a harmonious pattern 33 .In this study, we identified an elite wheat cultivar, GN29, which achieved harmonious improvement between multiple diseases resistance and a number of other elite traits.GN29 is an elite wheat cultivar with resistance to multiple important diseases.For powdery mildew resistance, it pyramided two elite Pm genes, Pm2 and Pm21, both of which have high breeding value.Pm2 was initially identified in the wheat landrace Ulka from the former Soviet Union in 1953 34 .Over the following 70 years, Pm2 was adequately used in wheat production and showed exceptional performance in fighting wheat powdery mildew 34 .To date, Pm2 still shows high and broad-spectrum resistance in some genetic backgrounds, such as the wheat cultivars/breeding lines 10 V-2, YingBo 700 and KM2939, and the landrace Niaomai 27,[36][37][38] .A similar situation occurs for Pm21, which is currently the most effective Pm gene and has been used in at least 20 cultivars 39 .Given the high value of Pm2 and Pm21, extending their operation lifespan during wheat production is imperative.Gene pyramiding is a promising strategy for developing durable resistance.Fortunately, Pm2 and Pm21 are pyramided in GN29 and achieve better collaboration, which is expected to be interdependent and can build durable resistance against continuous Bgt variations.In addition to powdery mildew resistance, GN29 also pyramided one Yr gene, Yr26, and two different kinds of Lr genes, Lr1 and Lr46.Among them, Yr26 is an elite resistance gene with high-and broad-spectrum resistance to stripe rust throughout the whole growth stage and has been used in production for many years 40 ; Lr1 is a frequently used resistance gene with leaf rust resistance throughout the whole growth stage 41 ; Lr46 is a resistance gene with slow resistance to leaf rust, and this locus is also resistant to stripe rust (Yr29) 42 , powdery mildew (Pm39) 43 , and stem rust (Sr58) 44 as a multiple resistant locus.Therefore, it is rare to pyramid so many resistance genes into a single cultivar, and meanwhile realize better collaboration.
Beyond the five different resistance genes, GN29 also pyramided two genes conferring drought tolerance, which implies its potential drought resistance ability and may be suitable for extension and application in arid and water-scarce regions.The Dreb gene is an important gene involved in abiotic stress tolerance in wheat production, including tolerance to drought, salinity, low temperature, and ABA 45 .The combination of Dreb and the Table 3. Seedling reaction patterns of Guinong 29 and several resistant stocks with documented powdery mildew resistance genes.Infection types (ITs) were scored according to the 0-4 scale, of which 0, 0; 1 and 2 were considered as resistant, while those with IT 3 and IT 4 were considered as susceptible.'-' represent missing data.www.nature.com/scientificreports/five resistance genes further suggests that GN29 may possess a cooperative ability to improve both biotic and abiotic stresses.GN29 also pyramided two low-molecular-weight glutenin subunit genes, Glu-B3b and Glu-B3bef, but no high-molecular-weight glutenin subunit genes, particularly the elite Dx5, were detected in GN29.Therefore, GN29 is regarded as a low-gluten cultivar and valuable for producing low-gluten flour, which is mainly used to make biscuits.

Table 4.
The presence/absence of different genes in Guinong 29 (GN29), Guinong 13 (GN13) and Guinong 21 (GN21).Given the extensive pyramiding of genes related to biotic and abiotic stress, as well as quality in GN29, an important question arises regarding the impact of these genes on agricultural and yield performance.From the agricultural and yield analyses, we found that GN29 maintained elite agricultural and yield performance and no obvious defects were observed.Meanwhile, GN29 can be considered a semi-winter cultivar based on the detection of vernalization genes [46][47][48] , suggesting that it is suitable for extension and application in southwestern wheat production regions, such as Guizhou, Yunnan, and Sichuang provinces of China, and in the south-central region of the northern winter wheat region.Agricultural and yield performances in distinct regions also indicated that GN29 has better adaptability, whether in Guizhou Province in the south or Hebei Province in the north.In addition, differences in plant height were obvious in different regions.Although three dwarfing genes were identified in GN29, they could not be adequately displayed in the Guizhou Province where GN29 has been selected and popularized.In Hebei Province, the three dwarfing genes were fully displayed, which may be related to different ecoclimatic conditions.

Conclusion
The wheat cultivar GN29 showed promising coordination between multiple diseases resistance and other key breeding traits.To determine the genetic foundation of these elite traits, GN29 was tested with 113 molecular markers for 98 target genes associated with diseases resistance, stress tolerance, quality, and adaptability.Several key genes were confirmed using genetic analysis, marker detection, and/or homology-based cloning.This study not only dissect the genetic basis of GN29 but also verified the harmonious improvement ability across multiple diseases resistance and other key breeding traits, which can provide elite gene resources and references for gene pyramiding during breeding practices.

Figure 4 .
Figure 4. Genomic in situ hybridization (GISH) (A) and nondenaturing FISH (ND-FISH) (B) analysis of Guinong 29 (GN29).(A) GISH analysis on the chromosome constitution in GN29 shows light blue hybridization signals evenly distributed by using Dasypyrum villosum genomic DNA as a probe, and the wheat chromosomes were counterstained with 4, 6-diamidino-2-phenylindole (DAPI) (dark blue).(B) ND-FISH analysis on the chromosome constitution of Guinong 29 shows no specific hybridization bands on chromosomes of the D. villosum in GN29.White arrows note the pair of added 6VS chromosomes.
Pm21, the diagnostic marker MBH1 of Pm21 was used to genotype the F 2:3 families phenotyped by Bgt isolate E18.